The mammary gland is a compound tubulo-alveolar gland that is composed of a series of branched ducts that, during lactation, drain sac-like alveoli (lobules). In the rodent, the mammary epithelium is embedded within a mammary fat pad rich in stromal (non-epithelial cells). The mammary epithelium is composed of two lineages of epithelial cells: the luminal cells (which make milk during lactation) and basal positioned myoepithelial cells. Generation and maintenance of the mammary epithelium is via the mammary stem cell (MaSC), which is defined here as that cell that can generate both the ductal and lobular structures of the mammary gland, can generate all the cell lineages of the mammary epithelium and can self-renew. The MaSC is of interest to the breast cancer biologist since cancer theory suggests that it is the stem cell, and possibly some of its more immediate descendants that have decreased stem cell potential but still have proliferative potential that are the targets for malignant transformation. As well, recent publications in the literature demonstrate that malignancies themselves have a stem cell component that propagates the tumor (Al-Hajj M, Wicha M S, Benito-Hernandez A, Morrison S J, Clarke M F. Proc Natl Acad Sci USA. 2003; 100:3983-8). This has huge implications in the treatment of cancer since it suggests that in order for cancer to be successfully contained or eradicated, it is the tumor stem cell component that has to be the therapeutic target. The ability to identify and purify mammary stem cells as well as mammary cells with high proliferative capacity but not necessarily having stem cell properties would be invaluable to the study of breast cancer and epithelial cell tumor biology.
In 1998 an experiment was performed which definitively demonstrated that a cell exists within the mouse mammary gland that fulfils the criteria of a MaSC (Kordon E C, Smith G H. Development 1998; 125:1921-30). Our own experiments involving transplantation of freshly isolated non-cultured mammary epithelial cells obtained from adult female mice into recipient mice indicates that MaSC occur at a frequency of about 1 cell in 1,300 total mammary cells and that there are approximately 1,400 MaSC per gland in the mouse.
There have been a number of in vitro studies trying to characterize the cells with proliferative potential in the human, mouse and rat mammary glands in a hope to identify the mammary stem cell. These experiments typically involve seeding phenotypically distinct subtypes of mammary cells at clonal densities in culture dishes in order to identify cells with growth potential by their ability to form colonies. Cells with the potential to form colonies in vitro are termed colony-forming cells (CFCs), and these assays detect all cells that have growth potential, regardless if they are stem cells or not. The inventors research data has demonstrated that the majority (>90%) of CFCs are not stem cells, but cells with growth potential that reside lower in the cellular hierarchy than stem cells. CFCs themselves can be subdivided into different subtypes such as luminal-restricted CFCs (which can only give rise to luminal cells) and bipotent CFCs (which can generate both luminal and myoepithelial cells). In the mouse mammary gland, approximately 90% of all CFCs are of the luminal-restricted type. The phenotypes of mammary CFCs isolated from different species are summarized in Table I.
These in vitro studies to characterize CFCs are limited because colony assays, in their current state, are unable to identify MaSC and to discriminate between MaSC that generate colonies from other CFCs that are deficient in stem cell properties.
The first insight into the phenotype of MaSC was reported by Welm and colleagues who demonstrated that expression of the cell surface molecule Sca-1 enriches for MaSCs that generate ductal-lobular outgrowths when transplanted into the cleared mammary fat pads of recipient mice (Welm B E, Tepera S B, Venezia T, Graubert T A, Rosen J M, Goodell M A. Dev Biol 2002; 245:42-56). However this is a crude enrichment strategy since approximately 20-60% of all mammary cells express Sca-1, and thus MaSC are far from being purified in Sca-1+ enriched subpopulations. To date, there has been no description of a method in the prior art that permits the isolation to high purity of stromal, luminal, myoepithelial, CFCs and MaSC subpopulations of mammary cells. The current method of invention satisfies this need.